One of the most promising approaches for understanding the molecular pathogenesis of cancer is to relate genetic changes, such as mutation or altered gene expression, to metastasis, treatment outcome, and survival, using high-throughput molecular biologic and proteomic methods. In cancers where the time between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations must be obtained with formalin-fixed paraffin-embedded (FFPE) tissues. However, large-scale multiplex techniques, such as proteomic analysis, serial analysis of gene expression, and gene chip methods using FFPE tissue have previously proven unsuccessful. The long-term goal of our research program is to improve public health by using high-throughput proteomic and molecular biologic screening methods to identify the molecular and genetic signatures of cancer. The objective of this proposal is to employ tissue surrogates to identify the formaldehyde-induced chemical modifications to proteins that occur during normal histologic tissue processing and to develop methods to reverse these modifications. Tissue surrogates are highly concentrated solutions of proteins that form tissue-like plugs following formaldehyde-induced intermolecular crosslinking. These plugs can be histologically processed like normal tissue. Our central hypothesis is that formaldehyde adducts and cross-links formed during histologic tissue processing can be sequentially reversed by a carefully designed series of heating, dialysis, rehydration, and protein renaturation steps, carried out under appropriate solvation conditions. We have formulated this hypothesis on the basis of our strong preliminary data, which have shown that the reversal of formaldehyde-induced chemical changes to proteins is relatively facile in aqueous solutions but requires a different approach for tissue that has been dehydrated in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for high-throughput proteomic screening of FFPE tissues. This will improve practical interventions for the diagnosis, treatment, and prevention of cancer and will facilitate the development of therapeutic agents. Our studies are innovative because we have developed a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins that occur during histologic processing. At the completion of this project, it is our expectation to have established a comprehensive understanding of the protein modifications that occur during tissue histology, together with methods for optimally reversing these modifications. This knowledge should result in the ability to carry out proteomic analysis using FFPE tissue.